The objective of the proposed research is the testing and development of a new concept for inhibiting HIV protease. Since this enzyme is critical for the replication of HIV, the causative agent of AIDS, and because its structure is unique to the virus it is an excellent target for anti-HIV agents. The dimeric nature of HIV protease makes possible a unique mode of enzyme inhibition. Prevention of HIV protease homodimer assembly or disruption of the assembled dimer interface would effectively block viral protease catalysis. Agents designed to inhibit dimerization of the protease, therefore, would disrupt the processing of essential viral proteins which would stop viral replication. The designed inhibitors mimic the structure of the terminal regions of HIV-l protease within one agent. These agents are unique in that the amino termini of peptides from the N- and C- terminal region of the protease are covalently crosslinked to yield a small HIV protease surrogate. The agents contain more than 50% of the residues needed for dimerization, but do not contain active site residues. Initial studies with crosslinked agents containing both the N- and C- terminal peptide sequences of HIV-1 protease have provided promising inhibitory data. These inhibitors will be improved upon using new agents which address questions such as: l) increased rigidity in the crosslinker, 2) increased water solubility, 3) increased interactions with the protease, 4) increase cell permeability, and 5) irreversible inhibition. When we have proven the concept of dimerization inhibition to be a valid one by obtaining sub-micromolar inhibitors of HIV-l protease with efficacy in the in vitro cell systems, our long term work will focus on issues such as in vivo stability and bioavailability which are critical for the production of a potent HIV therapeutic. Specific aims include: 1. Synthesize dimerization inhibitors of HIV-l protease based on crosslinked N- and C-terminal peptide sequences which address questions such as: a) increased rigidity in the crosslinker, b) increased water solubllity of the agents, c) increased interactions with the protease, d) increased cell permeability. 2. Synthesize irreversible dimerization inhibitors of the protease which covalently link the inhibitors to Cys95 of the protease through a maleimide moiety. 3. Evaluate HIV-l protease inhibition by the above agents. 4. Determine the mode of interaction of these agents with HIV- l protease through crosslinking, refolding and crystallization experiments. 5. Evaluate the in vitro activity of these agents against a variety of target cells and virus strains. 6. Synthesize new agents based on the above data to improve proteolytic stability.